Changes in cytosolic calcium ion concentrations ([Ca.sup.2+ ].sub.i) evoke a wide range of cellular responses. Intracellular Ca.sup.2+ -binding proteins are the key molecules in transducing Ca.sup.2+ signaling via enzymatic reactions or modulation of protein-protein interactions, some of which contribute to cell cycle events, and/or to cellular differentiation. Following stimulation of the cell by an external signal, second messenger molecules such as inositoltrisphosphate stimulate the brief release of [Ca.sup.2+ ].sub.i from the endoplasmic reticulum into the surrounding cytoplasm. Similar second messenger signaling pathways also occur in the dividing cell nucleus during breakdown of the nuclear membrane and segregation of chromatids during anaphase.
The calcium-binding domain of many proteins contains the high affinity Ca.sup.2+ -binding motif often referred to as the EF-hand. The EF-hand is characterized by a twelve amino acid residue-containing loop, flanked by two .alpha.-helices oriented approximately 90.degree. with respect to one another. Aspartate (D), and glutamate (E) or aspartate residues are usually found at positions 10 and 21, respectively, bordering the twelve amino acid loop. In addition, a conserved glycine residue in the central portion of the loop is found in most Ca.sup.2+ -binding EF-hand domains. Oxygen ligands within this domain coordinate the Ca.sup.2+ ion. Other non-EF-hand domain, Ca.sup.2+ -binding proteins (CBPs) bind Ca.sup.2+ using different protein conformations. (Kretsinger, R. H. and Nockolds, C. E. (1973) J. Biol. Chem. 248:3313-3326; and Celio, M. R. et al. (1996) Guidebook to Calcium-binding Proteins, Oxford University Press, Oxford, UK, pp. 15-20.)
Calmodulin (CaM) is the most widely distributed and the most common mediator of calcium effects. (Celio et al., supra pp. 34-40.) CaM appears to be the primary sensor of [Ca.sup.2+ ] changes in eukaryotic cells. The binding of Ca.sup.2+ to CaM induces marked conformational changes in the protein permitting interaction with, and regulation of over 100 different proteins. CaM interactions are involved in a multitude of cellular processes including, but not limited to, gene regulation, DNA synthesis, cell cycle progression, mitosis, cytokinesis, cytoskeletal organization, muscle contraction, signal transduction, ion homeostasis, exocytosis, and metabolic regulation.
CaM contains two pairs of EF-hand domains which are located in the N and C-halves of the molecule and connected by a flexible central helix. Binding of Ca.sup.2+ to the EF-hand domains of CaM induces a conformational change in the protein. In the presence of a target peptide, a further conformational change results in the flexible central helix being partially unwound and wrapped around the target peptide. In this manner, CaM interacts with a wide variety of target proteins. Several post-translational modifications of CaM including acylation of the amino terminus and phosphorylation of various serine and threonine residues have been reported.
The regulation of CBPs has implications for the control of a variety of disorders. Calcineurin, a CaM-regulated protein phosphatase, is a target for inhibition by the immunosuppressive agents cyclosporin and FK506. This indicates the importance of calcineurin and CaM in the immune response and immune disorders. (Schwaninger M. et al. (1993) J. Biol Chem. 268:23111-23115.) The level of CaM is increased several-fold in tumors and tumor-derived cell lines for various types of cancer. (Rasmussen, C. D. and Means, A. R. (1989) Trends in Neuroscience 12:433-438.)
The S100 proteins are a group of acidic Ca.sup.2+ -binding proteins with mass of approximately 10-12 kDa. These proteins are so named after the solubility of the first isolated protein in 100% saturated ammonium sulfate. The S100 proteins have two Ca.sup.2+ -binding domains. One domain is a low affinity Ca.sup.2+ -binding, basic helix-loop-helix site, the other domain is a high affinity Ca.sup.2+ -binding EF-hand type, acidic helix-loop-helix site. (Kligman, D. and Hilt, D. C. (1988) Trends Biochem. Sci. 13:437-442.) The EF-hand domain also encompasses a part of a region that specifically identifies members of the S100 family of proteins, but does not predict the Ca.sup.2+ -binding properties of the region. (See, e.g., SWISSPROT PROSITE pattern. accession number PS00303.) The distribution of particular S100 proteins is dependent on specific cell types, indicating that S100 proteins may be involved in transducing signals of increasing intracellular calcium in a cell type-specific fashion. For example, S100A13 protein is present in human and murine heart and skeletal muscle, and many other members of the S100 protein family, e.g., S100.beta. are abundant in brain. (Wu, T. et al. (1997) J. Biol. Chem. 272:17145-17153.)
Elevated serum levels of S100.beta. are associated with disseminated malignant melanoma metastases, suggesting that serum S100.beta. may be of value as a clinical marker for progression of metastatic melanoma. (Henze, G. et al. (1997) Dermatology 194:208-212.) Messenger RNA levels encoding both an S-100-like protein named calgizzarin and phospholipase A.sub.2 are elevated in colorectal cancers compared with those of normal colorectal mucosa. (Tanaka, M. et al. (1995) Cancer Lett. 89:195-200.)
The frequenin/neuronal calcium sensor protein from frog is a highly conserved protein that regulates rhodopsin phosphorylation and is found across a broad spectrum of phyla. It is present in the retina or other photosensitive organs of vertebrates, arthropods, molluscs, and nematodes; and in yeast. (De Castro, E. et al. (1995) Biochem. Biophys. Res. Comm. 216:133-140.)
Calcineurin homologous protein (CHP) and p22 are homologous CBPs which contain EF-hand motifs and show extensive protein sequence similarity to the regulatory subunit of protein phosphatase 2B, calcineurin B. (Lin, X. and Barber, D. L. (1996) Proc. Natl. Acad. Sci. 93: 12631-12636; and Barroso, M. R. et al. (1996) J. Biol. Chem. 271: 10183-10187.) CHP is widely expressed in human tissues. It specifically binds to and regulates the activity of NHE1, a ubiquitously expressed Na.sup.+ /H.sup.+ exchanger. Activation of NHE1 results in an increase in intracellular pH, which in turn activates cell proliferation, differentiation, and neoplastic transformation. The phosphorylation state of CHP is important for NHE1 regulation during cell division, and transient overexpression of CHP inhibits serum- and GTPase-stimulated NHE1 activities. (Lin and Barber, supra.) p22 is a cytosolic N-myristoylated phosphoprotein which undergoes conformational changes upon binding of calcium. p22 is ubiquitously expressed and may be required for regulating constitutive endocytic, membrane trafficking events. (Barroso et al., supra.)
Two CBPs associated with metaplasia and neoplasia are osteonectin and recoverin. Osteonectin is an anti-adhesive secreted glycoprotein involved in tissue remodeling and has one EF-hand, and a protein-protein or protein-heparin interaction domain. Recoverin was identified as an antigen in cancer-associated retinopathy, and is implicated in the pathway from retinal rod guanylate cyclase to rhodopsin. Recoverin is N-myristoylated at the N-terminus, and has three Ca.sup.2+ -binding sites including one low affinity Ca.sup.2+ -binding site and one high affinity Ca.sup.2+ -binding site. (Hohenester, E. et at. (1997) EMBO J. 16:3778-3786; and Murakami, A. et al. (1992) Biochem. Biophys. Res. Comm. 187:234-244.)
The discovery of new human calcium-binding proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of nervous, vesicle trafficking, developmental, neoplastic, immunological, and reproductive disorders.